Compounds that treat malaria and prevent malaria transmission

ABSTRACT

The invention provides methods and compounds for the treatment and prevention of malaria infection and transmission in a mammal by administering compounds of the invention to a mammal having or suspected of having a malaria infection. The invention also provides pharmaceutical compositions that can kill or arrest the growth of  Plasmodium  organisms, and especially  Plasmodium falciparum , thereby preventing or blocking transmission of malaria as well as treating malaria infection.

GOVERNMENT INTEREST

The work performed during the development of this disclosure utilizedintramural support from the National Institutes of Health. Thegovernment has certain rights in this invention.

TECHNICAL FIELD

The invention relates to therapeutic compounds, pharmaceuticalcompositions containing the same and their use in the prevention andtreatment of malaria infection.

BACKGROUND OF INVENTION

The life cycle of Plasmodium falciparum is one of the most complex lifecycles of any organism. The complete life cycle involves bothintracellular and extracellular stages in humans, as well as in themosquito.

Infection of a new human host is initiated when a carrier mosquito takesa blood meal. During the process of probing for food, saliva containingSporoziotes in the mosquito's saliva is injected into the human host.While some of these sporoziotes enter the bloodstream, recent work hasshown that a majority of them are deposited into the dermis and notdirectly into the blood. Within three hours following injection into theskin, most of the sporozoites leave the injection site via thebloodstream, the lymphatic system or direct migration through tissue.Regardless of the route taken, the sporozoite must migrate through cellbarriers in both the skin and in the ultimate target organ, the liver.To this end, it has recently been shown that Plasmodium sporozoites arecapable of traversing cells without initiating replication within thecell.

Once the sporozoites are in the bloodstream, they rapidly localize tothe liver. This preference for liver tissue is mediated by aninteraction between the major surface protein of sporozoites, thecircumsporozoite protein (CSP), and highly sulfated proteoglycanspresent in loose, basement membrane of the liver. Once they have reachedthe liver, it appears that sporozoites actively invade Kupffer cellsusing a process involving gliding motility. This process, which does notinvolve flagella, involves interactions between parasite membraneproteins and an extracellular, polysaccharide substrates secreted by theparasite. Additionally, cell invasion involves the ordered release ofproteins and other molecules from secretory organelles, calledmicronemes and rhoptries, present at the apical end of the zoite. Thesporozoite then traverse the invaded cell, crossing into the space ofDisse, and further migrating through several other hepatocytes. Thesporozoite then invades a final hepatocyte, with the parasite forming anencapsulating structure referred to as a parasitophorous vacuole (PV).At this point, the organism begins liver stage (LS) growth.

Little is known about growth of the organism during the liver stage.What is known, however, is that following formation of the PV, thesporozoite differentiates into a liver trophozoite. Following thisdifferentiation, growth and asexual replication through a process knownas exoerythrocytic schizogony is rapid, requiring the ability to obtainnutrients from the host, as well as the ability to cause an increase incell volume without damaging the host cell. This latter ability isrelated to the parasite's ability to confer resistance to apoptosis ofthe host cell. This stage of the life cycle culminates in the productionof mreozoites, which are released into the blood.

Once in the blood, the parasites begin the blood stage, or erythrocyticstage of the life cycle. It is this stage of the infection that resultsin the pathology associated with malaria. This stage is also referred toas the ring stage, due to the ring-like morphology of the earlytrophozoite. During this phase, the merozoites invade erythrocytes andundergo a trophic period in which the parasite enlarges. Enlargement ofthe trophozoite results in active metabolism, which includes ingestionof host cytoplasm and proteolysis of hemoglobin. The trophic phase endswith multiple rounds of nuclear division resulting in the formation ofschizonts. These schizonts then bud off merozoites, which are releasedupon rupture of the infected erythrocyte. Once released, the merozoitesinfect new erythrocytes, and begin another round of blood-stagereplication.

During subsequent rounds of replication, some parasites switch from anasexual replication strategy to a sexual replication strategy. In thisreproduction strategy, some parasites differentiate into eithermacrogametocytes or microgametocytes. These gametocytes, which contain asingle nucleus, are large parasites that fill the entire erythrocyte.Following their release into the blood, the gametocytes are ingested bymosquitoes during the taking of a blood meal. This begins what isreferred to as the sporogonic cycle.

Once in the mosquitoe's stomach, the microgametes penetrate themacrogametes, resulting tin the formation of zygotes. Soon after zygoteformation, meiosis occurs and the spherical zygote transforms into anelongated motile cell called an ookinete. The ookinete uses its motileability to penetrate the matrix surrounding the blood meal, and traverseseveral layers of epithelial cells before exiting through the basal sideof the epithelium. Upon reaching the basal surface, the ookinete beginsits transformation into an oocyst.

Maturation of the oocyst is a long process, taking approximately 12days. During this process, the oocyst grows in size, eventually becoming50-60 μm in diameter. At some point during this phase, sporozoites areproduced. These sporozoites are mobile, although this motility is stillimmature. Eventually, the sporozoites are released from the oocyst, duein large part to the action of a cysteine protease called the egresscysteine protease (ECP1).

Once released, the sporozoites are carried to all tissues of themosquito by the circulating hemolymph. Upon reaching the basal lamina ofthe salivary glands, ligands on the outer surface of the sporozoiteinteract with receptors, allowing the sporozoite to adhere to the basalsurface. The parasites move through the basal lamina and invade thesalivary gland acinar cells. This invasion is mediated by a short-livedvacuole that transports the sporozoite through the cytoplasm of theacinar cells, and out through the apical surface. The process ends withthe sporozoite ending up in the salivary gland duct. Thus, thesporozoites are ready to infect a new host during the next blood meal.

According to the Centers for Disease Control, malaria is one of the mostsevere public health problems worldwide, it is the leading cause ofdeath and disease in many developing countries, affecting mostly youngchildren and pregnant women. Malaria is the cause of at least onemillion deaths every year, with 350 and 500 million clinical episodesoccurring every year. More than 80% of the malaria deaths worldwideoccur in Africa south of the Sahara. Currently there is no vaccineavailable, and there is growing resistance to existing anti-malarialdrugs. Only one drug (primaquine) is used to kill non-erythrocyticstages (the gametocyte and liver stages), it has serious side effectsand the concern of resistance to the only drug that can killnon-erythrocytic stages prevents wide use of this drug. Thus, there isan urgent need for new malarial drugs and particularly drugs that caneffectively treat non-erythrocytic stages thereby disrupting theinfectious cycle of the infective organisms to prevent transmissionbetween individuals during the non-infectious gametocyte and liverstages and to eradicate the infection in an individual before theerythrocytic stage develops.

SUMMARY OF INVENTION

The present invention is drawn to compounds that can kill malariagametocytes to block malaria transmission and treat malaria infection inthe non-erythrocytic stages, as well as therapeutic uses of thesemolecules to prevent or slow the transmission of Plasmodium organismsbetween mammals and eliminate or prevent infection in a mammal.

The present inventors have identified compounds effective for thetreatment and prevention of malaria transmission by integratingquantitative high-throughput screening (qHTS) with genetic mapping.

Screens of seven malaria parasite lines revealed a large number ofconsensus actives (active in both replicates or active in one replicateand inconclusive in the other), all of which inhibited parasite growth.1,279 compounds were tested and about 20% to 30% were active in mostlines except W2, where 40% were active, and D10 and Dd2, where 19% and15% were active, respectively. Of the hundreds of inhibitors identifiedfor each line, about 50% or more showed IC₅₀ values between 1 and 10 μMand 6% to 14% had IC₅₀ values less than 1 μM. There were 155 compoundsthat inhibited growth in all seven lines tested and 25 compounds wereidentified that inhibited proliferation in all parasite lines at lowerthan 2 μM IC₅₀. Some of those 25 compounds are well known antimalarialdrugs (such as quinacrine), while others have not previously been usedfor malaria prophylaxis or treatment.

Thus, the present invention provides compounds that can kill or arrestthe growth of Plasmodium organisms, and especially Plasmodiumfalciparum, thereby preventing or blocking transmission of malaria aswell as treating malaria infection, including in the non-erythrocyticstages. The present invention also provides pharmaceutical compositionscontaining these compounds. The invention also provides methods of usingthese compounds and pharmaceutical compositions to treat malariainfection in a subject and to prevent or slow the development of malariainfection, including the non-erythrocytic stages of the disease.

Thus, one embodiment of the invention is a method of treating a malariainfection in a mammal by administering to a mammal in need of suchtreatment, a therapeutically effective amount of at least one compoundof the invention.

One embodiment of the invention is a method of preventing a malariainfection by administering to a mammal in need of such treatment, atherapeutically effective amount of a compound of the invention thatblocks malaria transmission by killing Plasmodium organisms in thenon-erythrocytic stages of the parasite's life cycle. In a preferredembodiment, the administered compound kills the Plasmodium gametocytes.

Another embodiment of the invention is a method of treating oreliminating a malaria infection in a mammal by administering to a mammalharboring a Plasmodium organism in a non-erythrocytic stage or suspectedof having been infected with a Plasmodium organism, a therapeuticallyeffective amount of a compound of the invention that blocks malariatransmission by killing Plasmodium organisms in the non-erythrocyticstages of the parasite's life cycle and/or blocking oocyst formation inthe mosquito midgut. In a preferred embodiment, the administeredcompound kills the Plasmodium gametocytes. In these embodiments, thecompound of the invention may be administered in conjunction (eithersimultaneously or sequentially) with an “artemisinin compound” such asartemisinin, artesunate, artemether and dihydroartemisinin. Preferably,in these embodiments, the compound of the invention is administered inconjunction (either simultaneously or sequentially) with a “thirdtherapeutically-effective anti-malaria compound” such as at least one oflumefantrine, amodiaquine, mefloquine, sulfadoxine, and pyrimethamine.More preferably, in these embodiments, the compound of the invention isadministered with both an artemisinin compound and a thirdtherapeutically-effective anti-malaria compound to effectively treaterythrocytic stages as well as non-erythrocytic stages of malaria. Thus,with the three-drug combination, the artemisinin compound and/or thethird therapeutically-effective anti-malaria compound effectively treatsymptoms of malaria while a compound of the invention, preferablyketotifen, blocks transmission of the infecting Plasmodium organism.

In one aspect, the compounds of the invention that are useful in themethods of the present invention include at least one of anantihistamine, a tricyclic antidepressant, A serotonin receptorantagonist, a dihydrofolate reductase (DHFR) inhibitor, a Na+ channelblocker, a mast cell stabilizing agent, an endothelial nitric oxidesynthase inhibitor, a selective blocker of apamin-sensitive K+ channels,a folic acid antagonist, a muscarinic receptor antagonist, an inducer ofapoptosis, a K+ channel blocker, a known antimalarial, a monoamineoxidase inhibitor, an anti-amoebic, an inhibitor of amyloid “42 fibrilformation, an inhibitor of microtubule assembly, a selectiveacetylcholinesterase inhibitor, a modulator of M2 muscarinicacetylcholine receptor activity, a selective PDGF tyrosine kinasereceptor inhibitor, a CYP1A1 and DNA topoisomerase II inhibitor, aninhibitor of free radical lipid peroxidation, a DNA synthesis inhibitor,a calcineurin phosphatase inhibitor, an antineoplastic, a DNAtopoisomerase I inhibitor, a protonophoric anthelmintic, an adrenoceptorantagonist, a Ca2+ ionophore, a potassium-sparing diuretic—anantibiotic, an acetylcholinesterase inhibitor, a vasoconstrictor, and,an adrenoceptor blocker.

In a specific embodiment, the compounds of the invention that are usefulin the methods of the present invention include at least one of anantihistamine, a tricyclic antidepressant, a serotonin receptorantagonist, a dihydrofolate reductase (DHFR) inhibitor, and a Na+channel blocker.

In another aspect, the compounds of the invention that are useful in themethods of the present invention include at least one of ketotifen,dequalinum dichloride, doxipin, protyptiline, carbita pentane,MLS000708402-02, NCGC00163169-03, MLS000556883-02, MLS000556884-02,cryphoheptidine, cryphoheptidine, desloratadine, sumotil, desloratadine,quetiapine, quetiapine, amitriptyline, butriptyline, desipramine,doxepin, nortriptyline, rimipramine, amitriptylinoxide, butriptyline,clomipramine, dosulepin, dothiepin, doxepin, imipramine, imipraminoxide,lofepramine, trimipramine, desipramine, norpramin, pertofrane,nortriptyline, protriptyline, demexiptiline, dibenzepin, dimetacrine,iprindole melitracen, metapramine, nitroxazepine, noxiptiline,propizepine, quinupramine, amineptine, opipramol, tianeptine,cianopramine, cyanodothiepin, fluotracen, amoxapine, maprotiline,mianserin, mirtazapine, setiptiline, oxaprotiline, diphenhydramine,doxylamine, loratadine, desloratadine, fexofenadine, pheniramine,cetirizine, promethazine, chlorpheniramine, levocetirizine, quetiapine,meclizine, dimenhydrinate, cimetidine, famotidine, ranitidine,nizatidine, roxatidine, lafutidine, protriptyline, trimipramine,cyproheptadine, trifluoperazine, topotecan, doxorubicin, mitoxantrone,iclaprim, amiloride, benzamil, quinidine sulfate, quinine sulfate,quinacrine dihydrochloride , diphenyleneiodonium chloride, dequaliniumdichloride, para-fluoro-hexahydrosila-difenidol (p-FHHSiD), emetinedihydrochloride hydrate, pentamidine isethionate, dequalinium analog,paclitaxel, tyrphostin A9, ellipticine, mitoxantrone, cyclosporin A,idarubicin, (S)-(+)-camptothecin, niclosamide, propafenonehydrochloride, calcimycin, (S)-(−)-propafenone hydrochloride,2′-(4-Aminophenyl)-[2,5′-bi-1H-benzimidazol]-5-amine (Ro 90-7501),1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide (BW284c51),WB 64, U-83836 dihydrochloride, aminopterin, methotrexate, halofantrine,pyrimethamine, triamterene, trimethoprim,1,5-Bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide,mefloquine, artemisinin, and, dihydroergotamine methanesulfonate.

In a preferred aspect of the invention, the compounds of the inventionthat are useful in the methods of the present invention include acompound of Formula I:

wherein;

X is C, N or S; and n=1 or 2 (i.e. the A and C rings may be 5-7 memberedrings, the B ring may be a 6-8 membered ring, preferably the A and Crings are 6-membered rings and the B ring is a 7-membered ring, each ofthe A, B, and C rings may be cycloalkyl or aryl, preferably, the A and Crings are aryl and the B ring is cycloalkyl)

R₁, R₂, and R₃ are each, independently, H, carbonyl, NR₅R₆, halide, C₁₋₆alkyl, C₃₋₈ cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, or C₁₋₄ alkoxy,aryl, or C₁₋₆ alkyl optionally substituted with NR₅R₆, hydroxy,mercapto, halide, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₄ alkoxy, aryl,heteroaryl, or a combination thereof;

R₄ is H, NR₅R₆, halide, C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₄ alkenyl, C₂₋₄alkynyl, C₁₋₄ alkoxy, aryl, 4-cyclohexylidene-1-methylpiperidine,methylpropan-1-amine, N,N-dimethylpropan-1-amine,N,N,2-trimethylpropan-1-amine, 1-propyl-4-methylpiperazine, or C₁₋₆alkyl optionally substituted with hydroxy, mercapto, halide, C₁₋₆ alkyl,C₁₋₆ alkenyl, C₁₋₄ alkoxy, cyclo alkyl, heterocyclic, aryl, heteroaryl,or a combination thereof;

R₅ and R₆ are each, independently, H, C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, or C₁₋₄ alkoxy.

In another embodiment, the invention is a method of preventing a malariainfection in a mammal by administering to the mammal at least one of thecompounds of the invention.

In these methods, the compounds of the invention may be administered astheir pharmaceutically acceptable salts, pharmaceutically acceptablesolvates, tautomers, racemates, polymorphs, pure enantiomers,diastereoisomers, metabolites, prodrugs or N-oxides.

Additionally, one aspect of the invention is a pharmaceuticalcomposition containing one or more of the compounds of the inventionwith at least one pharmaceutically acceptable carrier. A related aspectof the invention is a pharmaceutical composition comprising at least oneprodrug of the therapeutic compounds of the invention, with at least onepharmaceutically acceptable carrier. A related aspect of the inventionis a pharmaceutical package comprising a pharmaceutical compositioncomprising therapeutically-effective amounts of at least one compound ofthe invention, optionally together with at least one pharmaceuticallyacceptable carrier. The pharmaceutical compositions may be administeredseparately, simultaneously or sequentially, with other compounds ortherapies used in the prevention, treatment, prevention of transmission,or amelioration of symptoms of, malaria.

Also provided herein are pharmaceutical kits containing a pharmaceuticalcomposition of at least one prodrug of the invention, optionallytogether with at least one pharmaceutically acceptable carrier;prescribing information and a container. The prescribing information maydescribe the administration, and/or use of these pharmaceuticalcompositions alone or in combination with other therapies used in theprevention, treatment or amelioration of malaria.

Also provided herein are methods for the prevention, treatment,prevention of transmission, or prophylaxis of malaria in a mammalcomprising administering to a mammal in need thereof therapeuticallyeffective amounts of any of the pharmaceutical compositions of theinvention, including, for example, the pharmaceutical compositionscomprising at least one prodrug of the compounds of the invention.

Other aspects of the invention will be set forth in the accompanyingdescription of embodiments, which follows and will be apparent from thedescription or may be learned by the practice of the invention. However,it should be understood that the following description of embodiments isgiven by way of illustration only since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art and are encompassed within thescope of this invention.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1A-1D, show in vivo gametocytocidal activities of TAA compounds.Mice infected with Plasmodium yoelii nigeriensis were treated with drugsin different concentrations for various time periods and were exposed tomosquitoes. Oocysts were counted from dissected midgut 9-10 days afterfeeding. FIG. 1A, the number of oocysts from mosquitoes fed on micetreated with different TAA compounds for 1 hour. CAR, carbita Pentane(100 mg/kg); CHL, chlorpromazine (30 mg/kg); DEQ, dequalinium dichloride(10 mg/kg); Dox, doxipin hydrochloride (30 mg/kg); Ima, Imipramine (100mg/kg), KET, ketotifen (100 mg/kg); Pro, protryptiline hydrochloride (10mg/kg). FIG. 1B, the number of oocysts from mosquitoes fed on micetreated with different concentrations of ketotifen (KET) for 1 hour,compared with those treated with standard anti-malarial drugs,primaquine (PQ, 5 and 10 mg/kg), arteminisinin (ART, 5 mg/kg) andchloroquine (CQ, 10 mg/kg). FIG. 1C, the number of oocysts frommosquitoes fed on mice treated with a single dose of 0.5 mg/kg or 0.1mg/kg of ketotifen (KET) for 1, 6 or 24 hours. FIG. 1D, the number ofoocysts from mosquitoes fed on mice treated with two doses of 0.5 mg/kgor 0.1 mg/kg (filled symbol) of ketotifen (KET) for 1, 6 or 24 hours.CON, no drug control.

DESCRIPTION OF EMBODIMENTS

The present inventors have identified targets of chemical compounds inmalaria parasites by integrating quantitative high-throughput screening(qHTS) with genetic mapping. Seven P. falciparum lines were tested,including parents of three genetic crosses, for their responses to 1,279bioactive compounds from the LOPAC collection of known bioactives(Sigma-Aldrich). Using progeny from a genetic cross and genetictransfection methods of allelic replacement, candidate genes wereidentified for three differential chemical phenotypes (DCPs) that showdistinct signature responses to compounds among a variety of parasiteisolates. These results showed that differential responses of smallmolecules between parasite lines is a reliable phenotype for exploringmolecular mechanisms of pharmacologic interest in malaria treatment andprevention.

Thus, the present invention is drawn to methods of preventing ortreating malaria infection, and particularly to killing Plasmodiumorganisms infecting a mammal in the erythrocyte and non-erythrocytestages by administering at least one of the compounds of the invention.The invention encompasses methods of preventing or limiting thetransmission of Plasmodium organisms from one mammal to another, by theadministration of at least one of the compounds of the invention to oneor more of the mammals.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complicationcommensurate with a reasonable benefit/risk ratio.

“Pharmaceutically-acceptable salts” refer to derivatives of thedisclosed compounds wherein the parent compound is modified by makingacid or base salts thereof. Examples of pharmaceutically acceptablesalts include, but are not limited to, mineral or organic acid salts ofbasic residues such as amines, or alkali or organic salts of acidicresidues such as carboxylic acids. Pharmaceutically-acceptable saltsinclude the conventional non-toxic salts or the quaternary ammoniumsalts of the parent compound formed, for example, from non-toxicinorganic or organic acids. Such conventional nontoxic salts includethose derived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic,ethane disulfonic, oxalic, isethionic, and the like. Pharmaceuticallyacceptable salts are those forms of compounds, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

Pharmaceutically-acceptable salt forms of compounds provided herein aresynthesized from the parent compound which contains a basic or acidicmoiety by conventional chemical methods. Generally, such salts are, forexample, prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, nonaqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are preferred. Lists of suitable salts arefound in at page 1418 of Remington's Pharmaceutical Sciences, 17th ed.,Mack Publishing Company, Easton, Pa., 1985.

“Prodrugs” are intended to include any covalently bonded carriers thatrelease an active parent drug of the present invention in vivo when suchprodrug is administered to a mammalian subject. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (i.e.,solubility, bioavailability, half life, manufacturing, etc.) thecompounds of the present invention may be delivered in prodrug form.Thus, the present invention is intended to cover prodrugs of thepresently claimed compounds, methods of delivering the same, andcompositions containing the same. Prodrugs of the present invention areprepared by modifying functional groups present in the compound in sucha way that the modifications are cleaved, either in routine manipulationor in vivo, to a compound of the invention. Prodrugs include compoundsof the present invention wherein an acyl, hydroxy, amino, or sulfhydrylgroup is bonded to any group that, when the prodrug of the presentinvention is administered to a mammalian subject, is cleaved to form afree acetyl, hydroxyl, free amino, or free sulfydryl group,respectively. Examples of prodrugs include, but are not limited to,acetate, formate, and benzoate derivatives of alcohol and aminefunctional groups in the compounds of the present invention.

The term “therapeutically effective amount” of a compound of thisinvention means an amount effective to modulate the formation orprogression of a malaria infection in a host.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in, and may be isolated in,optically active and racemic forms. It is to be understood that thecompounds of the present invention encompasses any racemic,optically-active, regioisomeric or stereoisomeric form, or mixturesthereof, which possess the therapeutically useful properties describedherein. It is well known in the art how to prepare optically activeforms (for example, by resolution of the racemic form byrecrystallization techniques, by synthesis from optically-activestarting materials, by chiral synthesis, or by chromatographicseparation using a chiral stationary phase). It is also to be understoodthat the scope of this invention encompasses not only the variousisomers, which may exist but also the various mixtures of isomers, whichmay be formed. For example, if the compound of the present inventioncontains one or more chiral centers, the compound can be synthesizedenantioselectively or a mixture of enantiomers and/or diastereomers canbe prepared and separated. The resolution of the compounds of thepresent invention, their starting materials and/or the intermediates maybe carried out by known procedures, e.g., as described in the fourvolume compendium Optical Resolution Procedures for Chemical Compounds:Optical Resolution Information Center, Manhattan College, Riverdale,N.Y., and in Enantiomers, Racemates and Resolutions, Jean Jacques, AndreCollet and Samuel H. Wilen; John Wiley & Sons, Inc., New York, 1981,which is incorporated in its entirety by this reference. Basically, theresolution of the compounds is based on the differences in the physicalproperties of diastereomers by attachment, either chemically orenzymatically, of an enantiomerically pure moiety resulting in formsthat are separable by fractional crystallization, distillation orchromatography.

The compounds used in making the pharmaceutical compositions of thepresent invention may be purchased commercially. Additionally, some ofthe compounds of the invention may be purchased commercially. Thecompounds of the present invention, including the salts and prodrugs ofthese compounds, may also be prepared in ways well known to one skilledin the art of organic synthesis. These compounds of this invention maybe prepared using the reactions performed in solvents appropriate to thereagents and materials employed and suitable for the transformationbeing effected. It is understood by one skilled in the art of organicsynthesis that the functionality present on various portions of themolecule must be compatible with the reagents and reactions proposed.Such restrictions to the substituents, which are compatible with thereaction conditions, will be readily apparent to one skilled in the artand alternate methods must then be used.

Also provided herein are pharmaceutical compositions containingcompounds of the invention and a pharmaceutically-acceptable carrier,which are media generally accepted in the art for the delivery ofbiologically active agents to animals, in particular, mammals.Pharmaceutically-acceptable carriers are formulated according to anumber of factors well within the purview of those of ordinary skill inthe art to determine and accommodate. These include, without limitation:the type and nature of the active agent being formulated; the subject towhich the agent-containing composition is to be administered; theintended route of administration of the composition; and, thetherapeutic indication being targeted. Pharmaceutically-acceptablecarriers include both aqueous and non-aqueous liquid media, as well as avariety of solid and semi-solid dosage forms. Such carriers can includea number of different ingredients and additives in addition to theactive agent, such additional ingredients being included in theformulation for a variety of reasons, e.g., stabilization of the activeagent, well known to those of ordinary skill in the art. Descriptions ofsuitable pharmaceutically-acceptable carriers, and factors involved intheir selection, are found in a variety of readily available sources,such as Remington's Pharmaceutical Sciences, 17th ed., Mack PublishingCompany, Easton, Pa., 1985.

This invention further provides a method of treating a mammal afflictedwith a malaria infection or preventing a mammal from developing malaria,which includes administering to the mammal a pharmaceutical compositionprovided herein. Such compositions generally comprise a therapeuticallyeffective amount of a compound provided herein, that is, an amounteffective to prevent, ameliorate, lessen or inhibit a malaria infectionin a mammal. Such amounts typically comprise from about 0.1 to about1000 mg of the compound per kilogram of body weight of the mammal towhich the composition is administered. Therapeutically effective amountscan be administered according to any dosing regimen satisfactory tothose of ordinary skill in the art.

Administration may be, for example, by various parenteral means.Pharmaceutical compositions suitable for parenteral administrationinclude various aqueous media such as aqueous dextrose and salinesolutions; glycol solutions are also useful carriers, and preferablycontain a water soluble salt of the active ingredient, suitablestabilizing agents, and if necessary, buffering agents. Antioxidizingagents, such as sodium bisulfite, sodium sulfite, or ascorbic acid,either alone or in combination, are suitable stabilizing agents; alsoused are citric acid and its salts, and EDTA. In addition, parenteralsolutions can contain preservatives such as benzalkonium chloride,methyl- or propyl-paraben, and chlorobutanol.

Alternatively, compositions can be administered orally in solid dosageforms, such as capsules, tablets and powders; or in liquid forms such aselixirs, syrups, and/or suspensions. Gelatin capsules can be used tocontain the active ingredient and a suitable carrier such as, but notlimited to, lactose, starch, magnesium stearate, stearic acid, orcellulose derivatives. Similar diluents can be used to make compressedtablets. Both tablets and capsules can be manufactured as sustainedrelease products to provide for continuous release of medication over aperiod of time. Compressed tablets can be sugar-coated or film-coated tomask any unpleasant taste, or used to protect the active ingredientsfrom the atmosphere, or to allow selective disintegration of the tabletin the gastrointestinal tract.

A preferred formulation of the invention is a mono-phasic pharmaceuticalcomposition suitable for parenteral or oral administration for theprevention, treatment or prophylaxis of a malaria infection, consistingessentially of a therapeutically-effective amount of at least onecompound of the invention, and a pharmaceutically acceptable carrier. Ina preferred embodiment, the invention is a mono-phasic pharmaceuticalcomposition suitable for parenteral or oral administration for theprevention, treatment or prophylaxis of a malaria infection, consistingessentially of a therapeutically-effective amount of ketotifen, and apharmaceutically acceptable carrier. In another preferred embodiment,the invention is a mono-phasic pharmaceutical composition suitable forparenteral or oral administration for the prevention, treatment orprophylaxis of a malaria infection, consisting essentially of atherapeutically-effective amount of ketotifen and an artemisinincompound, and a pharmaceutically acceptable carrier. In anotherpreferred embodiment, the invention is a mono-phasic pharmaceuticalcomposition suitable for parenteral or oral administration for theprevention, treatment or prophylaxis of a malaria infection, consistingessentially of a therapeutically-effective amount of ketotifen, anartemisinin compound, and a third therapeutically-effective anti-malariacompound, in a pharmaceutically acceptable carrier. In theseembodiments, the “artemisinin compounds” are selected from artemisinin,artesunate, artemether and dihydroartemisinin. In these embodiments, the“third therapeutically-effective anti-malaria compound” is preferably atleast one of lumefantrine, amodiaquine, mefloquine, sulfadoxine, andpyrimethamine. In these embodiments, the artemisinin compound and/or thethird therapeutically-effective anti-malaria compound effectively treaterythrocytic stages and a compound of the invention, preferablyketotifen, effectively treat non-erythrocytic stages of malaria. Thus,with the three-drug combination, the artemisinin compound and/or thethird therapeutically-effective anti-malaria compound effectively treatsymptoms of malaria while a compound of the invention, preferablyketotifen, blocks transmission of the infecting Plasmodium organism.

Another preferred formulation of the invention is a mono-phasicpharmaceutical composition suitable for parenteral or oraladministration for the prevention, treatment or prophylaxis of a malariainfection in a mammal consisting essentially of atherapeutically-effective amount of a prodrug of at least one of thecompounds of the invention, and a pharmaceutically acceptable carrier.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as wetting agents,emulsifying agents and dispersing agents. It may also be desirable toinclude isotonic agents, such as sugars, sodium chloride, and the likein the compositions. In addition, prolonged absorption of the injectablepharmaceutical form may be brought about by the inclusion of agentswhich delay absorption such as aluminum monosterate and gelatin.

In some cases, in order to prolong the effect of a drug, it is desirableto slow the absorption of the drug from subcutaneous or intramuscularinjection. This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material having poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally-administered drug isaccomplished by dissolving or suspending the drug in an oil vehicle.

Injectable depot forms are made by forming microencapsule matrices ofthe drug in biodegradable polymers such as polylactide-polyglycolide.Depending on the ratio of drug to polymer, and the nature of theparticular polymer employed, the rate of drug release can be controlled.Examples of other biodegradable polymers include poly(orthoesters) andpoly(anhydrides). Depot injectable formulations are also prepared byentrapping the drug in liposomes or microemulsions which are compatiblewith body tissue. The injectable materials can be sterilized forexample, by filtration through a bacterial-retaining filter.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acompound of the present invention. When referring to thesepreformulation compositions as homogeneous, it is meant that the activeingredient is dispersed evenly throughout the composition so that thecomposition may be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, 0.1 to about 500 mg of the therapeuticcompounds of the present invention.

Formulations of the invention suitable for oral administration may be inthe form of capsules, cachets, pills, tablets, powders, granules or as asolution or a suspension in an aqueous or non-aqueous liquid, or anoil-in-water or water-in-oil liquid emulsions, or as an elixir or syrup,or as pastilles (using an inert base, such as gelatin and glycerin, orsucrose and acacia), and the like, each containing a predeterminedamount of a compound or compounds of the present invention as an activeingredient. A compound or compounds of the present invention may also beadministered as bolus, electuary or paste.

In solid dosage forms of the invention for oral administration(capsules, tablets, pills, dragees, powders, granules and the like), theactive ingredient is mixed with one or more pharmaceutically acceptablecarriers, such as sodium citrate or dicalcium phosphate, and/or any ofthe following: (1) fillers or extenders, such as starches, lactose,sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as,for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol;(4) disintegrating agents, such as agar-agar, calcium carbonate, potatoor tapioca starch, alginic acid, certain silicates, and sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as, for example, cetyl alcohol and glycerolmonosterate; (8) absorbents, such as kaolin and bentonite clay; (9)lubricants, such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and(10) coloring agents. In the case of capsules, tablets and pills, thepharmaceutical compositions may also comprise buffering agents. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules using such excipients as lactose or milksugars, as well as high molecular weight polyethylene glycols and thelike.

A tablet may be made by compression or molding optionally with one ormore accessory ingredients. Compressed tablets may be prepared usingbinder (for example, gelatin or hydroxypropylmethyl cellulose),lubricant, inert diluent, preservative, disintegrant (for example,sodium starch glycolate or cross-linked sodium carboxymethyl cellulose),surface-active or dispersing agent. Molded tablets may be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent.

The tablets, and other solid dosage forms of the pharmaceuticalcompositions of the present invention, such as dragees, capsules, pillsand granules, may optionally be scored or prepared with coatings andshells, such as enteric coatings and other coatings well known in thepharmaceutical-formulating art. They may also be formulated so as toprovide slow or controlled release of the active ingredient thereinusing, for example, hydroxypropylmethyl cellulose in varying proportionsto provide the desired release profile, other polymer matrices,liposomes and/or microspheres. They may be sterilized by, for example,filtration through a bacteria-retaining filter. These compositions mayalso optionally contain opacifying agents and may be of a compositionthat they release the active ingredient only, or preferentially, in acertain portion of the gastrointestinal tract, optionally, in a delayedmanner. Examples of embedding compositions which can be used includepolymeric substances and waxes. The active ingredient can also be inmicroencapsulated form.

The tablets or pills of the present invention may be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

Liquid dosage forms for oral administration of the compounds of theinvention include pharmaceutically-acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as, for example, water or other solvents,solubilizing agents and emulsifiers, such as ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propylene glycol, 1,3-butylene glycol, oils (in particular,cottonseed, groundnut, corn, germ, olive, castor and sesame oils),glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acidesters of sorbitan, and mixtures thereof.

Besides inert diluents, the oral compositions can also include adjuvantssuch as wetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents as, for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Formulations of the pharmaceutical compositions of the invention forrectal or vaginal administration may be presented as a suppository,which may be prepared by mixing one or more compounds of the inventionwith one or more suitable nonirritating excipients or carrierscomprising, for example, cocoa butter, polyethylene glycol, asuppository wax or salicylate, and which is solid at room temperature,but liquid at body temperature and, therefore, will melt in the rectumor vaginal cavity and release the active compound. Formulations of thepresent invention which are suitable for vaginal administration alsoinclude pessaries, tampons, creams, gels, pastes, foams or sprayformulations containing such carriers as are known in the art to beappropriate.

Dosage forms for the topical or transdermal administration of compoundsof this invention include powders, sprays, ointments, pastes, creams,lotions, gels, solutions, patches, drops and inhalants. The activeingredient may be mixed under sterile conditions with apharmaceutically-acceptable carrier, and with any buffers, orpropellants which may be required.

The ointments, pastes, creams and gels may contain, in addition to anactive ingredient, excipients, such as animal and vegetable fats, oils,waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc and zincoxide, or mixtures thereof.

Powders and sprays can contain, in addition to an active ingredient,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates and polyamide powder or mixtures of these substances.Sprays can additionally contain customary propellants such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Transdermal patches have the added advantage of providing controlleddelivery of compounds of the invention to the body. Such dosage formscan be made by dissolving, dispersing or otherwise incorporating one ormore compounds of the invention in a proper medium, such as anelastomeric matrix material. Absorption enhancers can also be used toincrease the flux of the compound across the skin. The rate of such fluxcan be controlled by either providing a rate-controlling membrane ordispersing the compound in a polymer matrix or gel.

Pharmaceutical formulations include those suitable for administration byinhalation or insufflation or for nasal or intraocular administration.For administration to the upper (nasal) or lower respiratory tract byinhalation, the compounds of the invention are conveniently deliveredfrom an insufflator, nebulizer or a pressurized pack or other convenientmeans of delivering an aerosol spray. Pressurized packs may comprise asuitable propellant such as dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation or insufflation, thecomposition may take the form of a dry powder, for example, a powder mixof one or more compounds of the invention and a suitable powder base,such as lactose or starch. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges, or, e.g.,gelatin or blister packs from which the powder may be administered withthe aid of an inhalator, insufflator or a metered-dose inhaler.

For intranasal administration, compounds of the invention may beadministered by means of nose drops or a liquid spray, such as by meansof a plastic bottle atomizer or metered-dose inhaler. Typical ofatomizers are the Mistometer (Wintrop) and Medihaler (Riker).

Drops, such as eye drops or nose drops, may be formulated with anaqueous or nonaqueous base also comprising one or more dispersingagents, solubilizing agents or suspending agents. Liquid sprays areconveniently delivered from pressurized packs. Drops can be delivered bymeans of a simple eye dropper-capped bottle or by means of a plasticbottle adapted to deliver liquid contents dropwise by means of aspecially shaped closure.

The formulations may be presented in unit-dose or multi-dose sealedcontainers, for example, ampules and vials, and may be stored in alyophilized condition requiring only the addition of the sterile liquidcarrier, for example water for injection, immediately prior to use.Extemporaneous injection solutions and suspensions may be prepared fromsterile powders, granules and tablets of the type described above.

The dosage formulations provided by this invention may contain thetherapeutic compounds of the invention, either alone or in combinationwith other therapeutically active ingredients, and pharmaceuticallyacceptable inert excipients. The term ‘pharmaceutically acceptable inertexcipients’ includes at least one of diluents, binders,lubricants/glidants, coloring agents and release modifying polymers.

Suitable antioxidants may be selected from amongst one or morepharmaceutically acceptable antioxidants known in the art. Examples ofpharmaceutically acceptable antioxidants include butylatedhydroxyanisole (BHA), sodium ascorbate, butylated hydroxytoluene (BHT),sodium sulfite, citric acid, malic acid and ascorbic acid. Theantioxidants may be present in the dosage formulations of the presentinvention at a concentration between about 0.001% to about 5%, byweight, of the dosage formulation.

Suitable chelating agents may be selected from amongst one or morechelating agents known in the art. Examples of suitable chelating agentsinclude disodium edetate (EDTA), edetic acid, citric acid andcombinations thereof. The chelating agents may be present in aconcentration between about 0.001% and about 5%, by weight, of thedosage formulation.

The dosage form may include one or more diluents such as lactose, sugar,cornstarch, modified cornstarch, mannitol, sorbitol, and/or cellulosederivatives such as wood cellulose and microcrystalline cellulose,typically in an amount within the range of from about 20% to about 80%,by weight.

The dosage form may include one or more binders in an amount of up toabout 60% w/w. Examples of suitable binders include methyl cellulose,hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone, eudragits, ethyl cellulose, gelatin, gum arabic, polyvinylalcohol, pullulan, carbomer, pregelatinized starch, agar, tragacanth,sodium alginate, microcrystalline cellulose and the like.

Examples of suitable disintegrants include sodium starch glycolate,croscarmellose sodium, crospovidone, low substituted hydroxypropylcellulose, and the like. The concentration may vary from 0.1% to 15%, byweight, of the dosage form.

Examples of lubricants/glidants include colloidal silicon dioxide,stearic acid, magnesium stearate, calcium stearate, talc, hydrogenatedcastor oil, sucrose esters of fatty acid, microcrystalline wax, yellowbeeswax, white beeswax, and the like. The concentration may vary from0.1% to 15%, by weight, of the dosage form.

Release modifying polymers may be used to form extended releaseformulations containing the therapeutic compounds of the invention. Therelease modifying polymers may be either water-soluble polymers, orwater insoluble polymers. Examples of water-soluble polymers includepolyvinylpyrrolidone, hydroxy propylcellulose, hydroxypropylmethylcellulose, vinyl acetate copolymers, polyethylene oxide,polysaccharides (such as alginate, xanthan gum, etc.), methylcelluloseand mixtures thereof. Examples of water-insoluble polymers includeacrylates such as methacrylates, acrylic acid copolymers; cellulosederivatives such as ethylcellulose or cellulose acetate; polyethylene,and high molecular weight polyvinyl alcohols.

Another embodiment of the invention relates to the use of any of theprodrug compounds or compositions described herein in the preparation ofa medicament for the treatment or prevention of malaria or transmissionof malaria.

Another embodiment of the invention relates to the use of any of thecompounds or compositions described herein in the preparation of amedicament for the prevention or treatment of malaria in a mammal. Theprevention may include the prevention of transmission of malaria betweenmammals.

Each publication or patent cited herein is incorporated herein byreference in its entirety.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLES Example 1 Screening of Bioactive Compounds using a PlasmodiumProliferation Assay

P. falciparum proliferation was tested within infected erythrocytesagainst the LOPAC-1280 collection of known bioactives (Sigma-Aldrich:sigmaaldrich.com/chemistry/drug-discovery/validation-libraries/lopac1280-navigator.html)by a qHTS 10 of a SYBR DNA binding assay (Plouffe D, et al. In silicoactivity profiling reveals the mechanism of action of antimalarialsdiscovered in a high-throughput screen. Proc Natl Acad Sci USA 2008;105:9059-64.). The P. falciparum lines used in this study have beendescribed previously (Mu J, et al. Multiple transporters associated withmalaria parasite responses to chloroquine and quinine. Mol Microbiol2003; 49:977-989). The SYBR Green viability assay was adapted frommethods described previously (Plouffe D, et al. In silico activityprofiling reveals the mechanism of action of antimalarials discovered ina high-throughput screen. Proc Natl Acad Sci USA 2008; 105:9059-64; KatoN, et al. Gene expression signatures and small-molecule compounds link aprotein kinase to Plasmodium falciparum motility. Nat Chem Biol 2008;4:347-56). Briefly, 3 μl culture medium was dispensed into 1536-wellblack clear-bottom plates (Aurora Biotechnologies) using a MultidropCombi (Thermo Fisher Scientific Inc.); 23-nL compounds in DMSO wereadded by a pin tool (Kalypsys), and 5 μl of P. falciparum-infected RBCs(0.3% parasitemia, 2.5% hematocrit final concentration) were added. Theplates were incubated at 37° C. in a humidified incubator in 5% CO2 for72 h, and 2 μl lysis buffer (20 mM Tris-HCl, 10 mM EDTA, 0.16% saponin,1.6% triton-X, 10× SYBR Green I supplied as 10,000× final concentrationby Invitrogen) was added to each well. The plates were mixed for 25 secwith gentle shaking and incubated overnight at room temperature in thedark. The following morning, fluorescence intensity at 485(14) nmexcitation and 535 (25) nm emission wavelengths was measured on anEnVision (Perkin Elmer) plate reader. The LOPAC₁₂₈₀ collection wasscreened against each line at eight (seven for Dd2) five-fold dilutionsbeginning at 29 μM. Antimalarial drugs, 2 and 3, and DMSO were includedas positive and negative controls for each plate, respectively.Follow-up SYBR Green assays in 96-well plate format were performed asdescribed (Liu S, Mu J, Jiang H, Su X.-z. Effects of Plasmodiumfalciparum mixed infections on in vitro antimalarial drug tests andgenotyping. Am J Trop Med Hyg 2008; 79:178-84). Briefly, 150 μlsynchronized parasites diluted to 1% parasitemia with 1% hematocrit weremixed with 50 μl medium containing compound. Compound stocks (10 mM)were dissolved in ethanol or DMSO and tested at 11 two-fold dilutions intriplicate. The beginning concentration of each compound was adjusteddepending on IC₅₀ values from the initial qHTS. The plates wereincubated at 37° C. under 5% CO2, 5% O2, and 90% N2 for 72 h, incubatedfor another 30 min after addition of 50 μl lysis buffer, and read in aFLUOstar OPTIMA reader (BMG Labtech). Data were analyzed using softwareat the NIAID Bioinformatic Resources(niaid-biocluster.niaid.nih.gov/cgi-bin/bipod2/index.cgi). Parasiteproliferation was measured after 72 hr of incubation (corresponding to1.5 generations of intra-erythrocytic parasite growth), with eachcompound tested at seven or eight five-fold dilutions beginning at 29μM. Two independent screens of each parasite line performed well,showing 0.7 or higher average Z′ factor and eight-fold or highersignal-to background ratio. The potencies of known antimalarial agentshad similar values determined by the assay in 96-well plate format.Titrations of two control inhibitors were present on every plate andshowed expected IC₅₀ values. The antimalarial agents chloroquine andquinine were present in the collection, and IC₅₀ values determined fromthe qHTS for 5 were similar, but the measurements for 4 were 15- to20-fold higher than those from 96-well plate tests because of lowersolubility of 4 in dimethyl sulfoxide (DMSO). Although the determinedpotencies of 4 were lower, the relative potency between lines sensitiveto 4, HB3, 3D7, and D10 were clearly distinguished from resistant lines.The consensus IC₅₀ and activity values for each of the 1,279 compoundswere established and screening data are deposited in PubChem (AID 1828).Comparison of the replicate runs for each parasite line indicatedexcellent agreement of curve class assignment and IC₅₀ determination.About 80% of actives identified in one replicate were active in thesecond replicate for all lines except Dd2, where 55% were active in bothreplicates. Of the actives that did not repeat, almost all showedinconclusive activity in the other replicate with few or none scoring asinactive. The potencies of compounds scored as active or inconclusive inboth replicates correlated well indicating good repeatability indetermining IC₅₀ values between replicates.

Example 2 Discovery of Potential Antimalarial Compounds

Screens of the seven parasite lines revealed a large number of consensusactives (active in both replicates or active in one replicate andinconclusive in the other), all of which inhibited parasite growth.Among the 1,279 compounds tested, about 20% to 30% were active in mostlines except W2, where 40% were active, and D10 and Dd2, where 19% and15% were active, respectively. Of the hundreds of inhibitors identifiedfor each line, about 50% or more showed IC₅₀ values between 1 and 10 μMand 6% to 14% had IC₅₀ values less than 1 μM. There were 155 compoundsthat inhibited growth in all seven lines tested. The potencydistribution of these pan inhibitors indicated differences insensitivity between the lines; W2 was most sensitive, with 32% of thecompounds having IC₅₀ values of 1 μM or less, while Dd2 was leastsensitive with 7% below 1 μM. We identified 25 potent compounds thatinhibited proliferation in all parasite lines at lower than 2 μM IC₅₀.Some of these compounds are well known antimalarial drugs, while otherssuch as hexahydro-sila-difenidol hydrochloride, dequalinium dichloride,taxol, and BW 284c51 are not compounds used for malaria prophylaxis ortreatment.

Example 3 Discovery of Plasmodium Gametocytocidal Compounds

2,816 FDA approved drugs were screened against two Plasmodium falciparumparasites, one producing functional gametocytes and the other producingno gametocytes, and identified approximately 41 compounds that showeddifferences between the two parasites (5 folds differences in IC₅₀) inresponse to the drugs.

For testing transmission-blocking activity in vivo, CD-1 strain femalemice, aged 6-8 weeks old, were infected intra-peritoneally (i.p.) with5×106 P. yoelii nigeriensis. In a preliminary experiment, on day 3 postinfection the animals were administrated i.p. a single dose of tricyclicantihistamine and antidepressants (TAA) compound or pharmaceutical-gradephosphate buffer saline (pH 7.4). Starting 1 hr after the administrationof the TAA compound, the animals were anesthetized and brought intocontact with a colony of Anopheles stephensi mosquitoes. The mosquitoeswere allowed to feed on the mice. Unfed or partially engorged mosquitoeswere removed from the cages. The mosquitoes were maintained for 9-10days 24±1° C. and 80-90% relative humidity. The gametocytocidal activityof the test compound was accessed on the basis of the oocyst counts inmosquitoes fed on treated animals and a control group. In subsequentexperiments, the gametocytocidal action of ketotifin at differentconcentrations and doses was evaluated similarly. One group was given asingle dose of 0.1, 0.5, 1, 5, 10, 30 or 60 mg/kg on day 3 postinfection and subjected to mosquito feeding 1 hr after compoundadministration. Primaquine, chloroquine and arteminsin were used asreference drugs. The other group was given one does or two doses of 0.1or 0.5 mg/kg at 4 hr interval and subjected to mosquito feeding 1, 6 or24 hrs after the final drug administration.

Whereas mosquitoes fed on mice without drug treatment had an average of460 oocysts per mosquito, mosquitoes fed on mice treated with ketotifen(100 mg/kg) and protryptiline hydrochloride (10 mg/kg) showed a strongreduction in oocyst formation (FIG. 1A). In particular, ketotifendramatically reduced the number of oocysts to an average of 34 oocystsper mosquito at 1 mg/kg, and almost completely blocked oocystdevelopment at 5 mg/kg after treatment for 1 hr (FIG. 1B). The oocystcounts from 5 mg/kg ketotifen treatment group (0.13 oocysts permosquito; 2 of 44 mosquitoes had oocysts) were also significantly lowerthose of 5 mg/kg primaquine treatment group (6 oocysts per mosquito; 7of 21 mosquitoes had oocysts). Further reduction in oocyst counts wasalso obtained using a single dose of 0.5 and 0.1 mg/kg of ketotifen whentreatment times were extended to 24 hours prior to mosquito feeding (12and 82 oocysts per mosquito, respectively) (FIG. 1C). Two doses of 0.5mg/kg or 0.1 mg/kg each with 4 hours interval of drug administrationwere also tested (FIG. 1D). Whereas the oocyst counts in double-dosetreatment of 0.1 mg/kg with 24-hour treatment time (80 oocysts permosquito) were similar to those of single-dose treatment, double-dosetreatment of 0.5 mg/kg with 24-hour treatment almost completelyeliminated oocysts in the mosquitoes (2 oocysts per mosquito; 4 of 23mosquitoes had oocysts).

Compounds that showed differences in response between gametocyteproducing and non-producing parasites were effective in killinggametocytes. In particular, the compound ketotifen was very effective inblocking oocyst formation in mosquito, achieving similar reduction inoocyst count to primaquine using a dose that is 10% of that ofprimaquine. Ketotifen can also completely kill P. falciparum gametocytesin vitro at 0.5 μM. Similar tricyclic structures were tested in mice andin vitro, and many were found to be active in blocking oocyst formation.These compounds are drugs that can be used to block parasitetransmission.

The foregoing description of the present invention has been presentedfor purposes of illustration and description. Furthermore, thedescription is not intended to limit the invention to the form disclosedherein. Consequently, variations and modifications commensurate with theabove teachings, and the skill or knowledge of the relevant art, arewithin the scope of the present invention. The embodiments describedhereinabove are further intended to explain the best mode known forpracticing the invention and to enable others skilled in the art toutilize the invention in such, or other, embodiments and with variousmodifications required by the particular applications or uses of thepresent invention. It is intended that the appended claims be construedto include alternative embodiments to the extent permitted by the priorart.

What is claimed is:
 1. A method of blocking oocyst formation andtransmission of a Plasmodium parasite comprising administering to amammal in need of such treatment, a therapeutically-effective amount ofat least one compound selected from the group consisting of: a) acompound of Formula I:

wherein; X is C, N or S; and n=1 or 2 (i.e. the A and C rings may be 5-7membered rings, the B ring may be a 6-8 membered ring, preferably the Aand C rings are 6-membered rings and the B ring is a 7-membered ring,each of the A, B, and C rings may be cycloalkyl or aryl, preferably, theA and C rings are aryl and the B ring is cycloalkyl) R₁, R₂, and R₃ areeach, independently, H, carbonyl, NR₅R₆, halide, C₁₋₆ alkyl, C₃₋₈cycloalkyl, C₂₋₄ alkenyl, C₂₋₄ alkynyl, or C₁₋₄ alkoxy, aryl, or C₁₋₆alkyl optionally substituted with NR₅R₆, hydroxy, mercapto, halide, C₁₋₆alkyl, C₁₋₆ alkenyl, C₁₋₄ alkoxy, aryl, heteroaryl, or a combinationthereof; R₄ is H, NR₅R₆, halide, C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, C₁₋₄ alkoxy, aryl,4-cyclohexylidene-1-methylpiperidine, methylpropan-1-amine,N,N-dimethylpropan-1-amine, N,N,2-trimethylpropan-1-amine,1-propyl-4-methylpiperazine, or C₁₋₆ alkyl optionally substituted withhydroxy, mercapto, halide, C₁₋₆ alkyl, C₁₋₆ alkenyl, C₁₋₄ alkoxy, cycloalkyl, heterocyclic, aryl, heteroaryl, or a combination thereof; R₅ andR₆ are each, independently, H, C₁₋₆ alkyl, C₃₋₈ cycloalkyl, C₂₋₄alkenyl, C₂₋₄ alkynyl, or C₁₋₄ alkoxy, b) a compound that is at leastone of ketotifen, dequalinum dichloride, doxipin, protyptiline, carbitapentane, kitotifen, MLS000708402-02, NCGC00163169-03, MLS000556883-02,MLS000556884-02, primaquine, cryphoheptidine, cryphoheptidine,desloratadine, sumotil, desloratadine, quetiapine, quetiapine,amitriptyline, butriptyline, desipramine, doxepin, nortriptyline,rimipramine, amitriptylinoxide, butriptyline, clomipramine, dosulepin,dothiepin, doxepin, imipramine, imipraminoxide, lofepramine,trimipramine, desipramine, norpramin, pertofrane, nortriptyline,protriptyline, demexiptiline, dibenzepin, dimetacrine, iprindolemelitracen, metapramine, nitroxazepine, noxiptiline, propizepine,quinupramine, amineptine, opipramol, tianeptine, cianopramine,cyanodothiepin, fluotracen, amoxapine, maprotiline, mianserin,mirtazapine, setiptiline, oxaprotiline, diphenhydramine, doxylamine,loratadine, desloratadine, fexofenadine, pheniramine, cetirizine,promethazine, chlorpheniramine, levocetirizine, quetiapine, meclizine,dimenhydrinate, cimetidine, famotidine, ranitidine, nizatidine,roxatidine, lafutidine, protriptyline, trimipramine, cyproheptadine,trifluoperazine, topotecan, doxorubicin, mitoxantrone, pyrimethamine,iclaprim, amiloride, benzamil, quinidine sulfate, quinine sulfate,quinacrine dihydrochloride , diphenyleneiodonium chloride, dequaliniumdichloride, methotrexate, para-fluoro-hexahydrosila-difenidol(p-FHHSiD), emetine dihydrochloride hydrate, pentamidine isethionate,dequalinium analog, paclitaxel, tyrphostin A9, ellipticine,mitoxantrone, cyclosporin A, idarubicin, (S)-(+)-camptothecin,niclosamide, propafenone hydrochloride, calcimycin, (S)-(−)-propafenonehydrochloride, 2′-(4-Aminophenyl)[2,5′-bi-1H-benzimidazol]-5-amine (Ro90-7501), 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide(BW284c51), WB 64, U-83836 dihydrochloride, aminopterin, methotrexate,halofantrine, pyrimethamine, triamterene, trimethoprim,1,5-Bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide,mefloquine, artemisinin, and, dihydroergotamine methanesulfonate, and c)at least one of an antihistamine, a tricyclic antidepressant, Aserotonin receptor antagonist, a dihydrofolate reductase (DHFR)inhibitor, a Na+ channel blocker, a mast cell stabilizing agent, anendothelial nitric oxide synthase inhibitor, a selective blocker ofapamin-sensitive K+ channels, a folic acid antagonist, a muscarinicreceptor antagonist, an inducer of apoptosis, a K+ channel blocker, aknown antimalarial, a monoamine oxidase inhibitor, an anti-amoebic, aninhibitor of amyloid “42 fibril formation, an inhibitor of microtubuleassembly, a selective acetylcholinesterase inhibitor, a modulator of M2muscarinic acetylcholine receptor activity, a selective PDGF tyrosinekinase receptor inhibitor, a CYP1A1 and DNA topoisomerase II inhibitor,an inhibitor of free radical lipid peroxidation, a DNA synthesisinhibitor, a calcineurin phosphatase inhibitor, an antineoplastic, a DNAtopoisomerase I inhibitor, a protonophoric anthelmintic, an adrenoceptorantagonist, a Ca2+ ionophore, a potassium-sparing diuretic, anantibiotic, an acetylcholinesterase inhibitor, a vasoconstrictor, and,an adrenoceptor blocker.
 2. A method of treating malaria by killing orarresting the growth of Plasmodium organisms in a mammal, comprisingadministering at least one compound of claim 1 to a mammal in need ofsuch treatment.
 3. The method of claim 2, wherein the Plasmodiumorganism is in a non-erythrocytic stage.
 4. The method of claim 2,wherein the compound effectively kills Plasmodium gametocytes.
 5. Themethod of claim 1 or 2, wherein the at least one compound isadministered as a pharmaceutical composition.
 6. The method of claim 1or 2, wherein the at least one compound is administered as apharmaceutically acceptable salt, pharmaceutically acceptable solvate,tautomer, racemate, polymorph, pure enantiomer, diastereoisomer,metabolite, prodrug or N-oxide.
 7. The method of claim 1 or 2, whereinthe at least one compound is ketotifen.
 8. The method of claim 1 or 2,wherein the at least one compound is ketotifen and artemisinin.
 9. Themethod of claim 1 or 2, wherein the at least one compound is ketotifenand at least one drug selected from the group consisting of artemisinin,artesunate, artemether, dihydroartemisinin, lumefantrine, amodiaquine,mefloquine, sulfadoxine, and pyrimethamine.
 10. The method of claim 9,wherein the at least one compound and the at least one drug areadministered simultaneously.
 11. The method of claim 9, wherein the atleast one compound and the at least one drug are administeredsequentially.
 12. A mono-phasic pharmaceutical composition suitable forparenteral or oral administration for the prevention, treatment orprophylaxis of a malaria infection, consisting essentially of atherapeutically-effective amount of ketotifen, and a pharmaceuticallyacceptable carrier.
 13. A mono-phasic pharmaceutical compositionsuitable for parenteral or oral administration for the prevention,treatment or prophylaxis of a malaria infection, consisting essentiallyof a therapeutically-effective amount of ketotifen and artemisinin, anda pharmaceutically acceptable carrier.
 14. A mono-phasic pharmaceuticalcomposition suitable for parenteral or oral administration for theprevention, treatment or prophylaxis of a malaria infection, consistingessentially of a therapeutically-effective amount of ketotifen and atleast one drug selected from the group consisting of artemisinin,artesunate, artemether, dihydroartemisinin, lumefantrine, amodiaquine,mefloquine, sulfadoxine, and pyrimethamine, and a pharmaceuticallyacceptable carrier.